Project Summary/Abstract Patients with certain subtypes of leukemia are associated with dismal outcomes due to resistance to current treatment options, particularly for those with MLL rearrangements. Chronic NF-?B activity is observed in leukemia cells, especially within the leukemia stem cell (LSC) population, and is implicated as a requirement for leukemogenesis, including the MLL-driven leukemia. Given the pleotropic function of NF-?B, targeting the leukemia-specific function of NF-?B is urgently needed. However, the molecular mechanism of the leukemia- specific function of NF-?B is unclear. Previous studies implicate the adaptor protein Sequestosome 1 (also known as p62) as dispensable for normal hematopoietic stem and progenitor cell (HSPC) function. Intriguingly we find that p62 is overexpressed in leukemia cells, and associated with increased TNF? in leukemia patients. p62 was shown to form a signalosome with RIPK1 (p62/RIPK1) via its ZZ domain in response to TNF?, leading to NF-?B activation. Our functional study reveals that p62 is required for leukemia cell function through activating NF-?B, indicating HSPC acquire a dependency on p62 function during transformation and p62-mediated signaling pathway represents a leukemia-specific mechanism that activates NF-?B. My long-term goal is to improve the targeted therapy by identifying leukemia-specific signaling pathways and testing novel therapeutic approaches. The objectives of this proposal are to: (1) determine the contribution of p62 to promoting leukemia; (2) elucidate the molecular mechanism of p62 in activating NF-?B in leukemia; and (3) test targeting the leukemia-specific p62 signalosome with a small molecule compound as a means to inactive NF-?B and inhibit leukemia cell and LSC while preserving normal HSPC. We hypothesize that p62 promotes leukemia by forming a leukemia- specific p62/RIPK1 signalosome that activates NF-?B. In particular, given that p62 supports MLL leukemia cell growth and NF-?B mediates MLL-driven leukemogenesis, we will determine the contribution of p62 in promoting MLL leukemia by examining preleukemia and leukemia phenotype in Mll-AF9 knockin mice followed by p62 deletion (Mll-AF9+/-;p62????mice). We will determine whether p62 promotes leukemia through activating NF-?B. In addition, we will determine whether p62 binds RIPK1 in leukemia cells, and whether the p62/RIPK1 signalosome is essential for NF-?B activation and leukemogenesis. Moreover, we will examine whether the ZZ domain on p62 is required for forming the p62/RIP signalosome, activating NF-?B and promoting leukemia. Finally, we will test a small molecule compound that specifically targets the p62 ZZ domain in disrupting the p62/RIPK1 signalosome, inactivating NF-?B and inhibiting leukemia cells and LSC while preserving normal HSPC. We anticipate that targeting the leukemia-specific p62 signalosome exerts antileukemia effect without damaging normal cells. The proposed study will impact on our understanding on leukemia-specific signaling pathways. The findings from this study will help us design novel therapeutic strategies that improve the specificity of targeted therapy for aggressive subtypes of leukemia.